Human mtdna monitoring primer set

Cells were washed with clean PBS buffer and collected in fresh PBS buffer with cell scrapers. Genomic DNA including nuclear DNA and mtDNA was purified using the DNeasy Blood & Tissue Kit (QIAGEN) and treated with RNase A (QIAGEN) to remove RNA and Proteinase K (QIAGEN) to digest proteins for obtaining highly purified DNA samples. A relative mtDNA copy number was analyzed with Human mtDNA Monitoring Primer Set (Takara Bio). Genomic DNA (10 ng) was applied in the PCR system, and data analysis was performed following the manufacturer’s manual.

Human and mouse mtDNA and nuclear DNA was isolated from 50 million human iPS cells (clone A4) or 30 mg fresh liver tissue of C57BL/6 mice using Mitochondrial DNA Isolation Kit (abcam) following the manufacturer’s instruction. Human and mouse mtDNA was linearized by restriction digestion at unique sites using SphI-HF or MluI respectively, (New England Biolabs) and subjected to agarose gel purification to isolate the ~16.5 kbp DNA band. Purity of human mtDNA was evaluated by qPCR detection of mtDNA markers (MT-ND1 and MT-ND5) and nuclear DNA markers (SLCO2B1 and SERPINA1) using Human mtDNA Monitoring Primer Set (Cat# 7246; Takara Bio). Purity of mouse mtDNA was evaluated by qPCR quantitation of mtDNA marker MT-ND1 (Mm04225274-s1) and nuclear DNA marker tert1 (Cat# 4403316) using Taqman Assays (Life Technologies).